This proposal deals with the study of phospholipids in mammalian cells, and is focussed on the general areas of organization, dynamics, and metabolism of these components at the cell surface. We will continue to develop techniques for introduction of defined phospholipid molecules into the cell surface. Liposome-cell fusion and lipid transfer mechanisms will be exploited to selectively introduce exogenous phospholipids into either the inner or outer leaflets of the plasma membrane bilayer of intact cells. As an alternative approach, phospholipid exchange proteins which have been charged with radioactive lipids will also be used for the introduction of foreign lipids into cells. We will use these techniques to study the metabolism of cell surface phospholipids, and to determine if the inner and outer leaflets of the plasma membrane bilayer are processed differently. Such studies may have a direct bearing on the membrane biogenesis and on the origin and maintenance of lipid asymmetry. Antigenic phospholipids will be inserted into the surface membrane of mammalian cells, either using liposomes or by growing cells in the presence of antigenic lipid precursors. The organization and behavior of such lipids in the cells will then be studied using appropriate immunochemical techniques. Particular attention will be devoted toward studying the mechanisms of patching and capping of haptenated lipids in mammalian cells, a phenomenon already documented in preliminary experiments.